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Abstract

Cancer is one of the main reasons of death in the most countries and in Iran. Immunotherapy quickly became one of the best methods of cancer treatment, along with chemotherapy and radiation. “Immunotoxin Therapy” is a promising way of cancer therapy that is mentioned in this field. Immunotoxins are made from a toxin attaching to an antibody target proteins present on cancer cells. The first-generation immunotoxins were made of a full-length toxin attached to whole monoclonal antibodies. But, these immunotoxins could bind to normal cells. DAB389IL2 was the first immunotoxin approved by the Food and Drug Administration. Current trends and researches are ongoing on finding proteins that in combination with immunotoxins have minimal immunogenicity and the most potency for target cell killing.

Keywords

Toxins antibody immunotoxins cancer therapy

Introduction

The human immune system is essential to protect body against disease, but can be harmful under specific circumstances, including treatment of human diseases in specific conditions like cancer by use of foreign (i.e. nonhuman) proteins.¹ Cancer is one of the main reasons of death in the most developed countries, and it is the third main cause of death in Iran.² So far, various modern treatments for cancer such as chemotherapy, radiation, and surgery have not been able to reduce mortality rates completely. Surgical removing of solid tumors often fails due to disease recurrence. Chemotherapeutic drugs are non-specific and increasing the dosage causes resistance which may lead to peripheral toxicity. Radiotherapy has disadvantageous effects on normal cells in the body.³ For this reason, the researchers proposed a new idea for cancer therapy called “immunotoxins.” It was based on attaching a toxin to an antibody which targets proteins present only on cancer cell surface.⁴ Immunotherapy quickly became one of the best methods of cancer treatment, along with chemotherapy and radiation.⁵

History

In the early 1870s, Paul Ehrlich found that feeding modicum amounts of castor seeds induced immunity to mice. Evidence showed that specific neutralizing antibodies called “magic bullets” were produced under such conditions and could kill cancer cells.⁶ The primary functional immunotoxins were made of full-length antibodies coupled with plant toxins like ricin or gelonin without toxin’s binding domain.^7–9 Subsequently, bacterial protein toxins like Pseudomonas exotoxin (PE)¹⁰ and diphtheria toxin (DT)¹¹ were used.

The first-generation immunotoxins were made of a full-length PE protein attached to whole monoclonal antibodies. But, these immunotoxins could bind to normal cells. Loss of specificity, low stability, and heterogeneous composition were the flaws of the first-generation immunotoxins. Knowing the structure and function of toxins helped to design the second-generation immunotoxins that were made by chemical bonding methods. In that method, regions of the toxin that were not essential for cell killing were removed. The resulting toxin fragment was coupled to a specific antibody and could not bind to normal cells. Thus, PE38 was born—a small and truncated toxin which could be linked to cells by attaching to an antibody but could not bind to or kill cells when it is alone. In addition to the heterogeneity, large molecular size limited their entry into tumors, and some immunotoxins attached to normal cells and caused difficulties like vascular leak syndrome (VLS), hemolytic uremic syndrome, and pleuritis.

Those side effects were reduced in the next design of immunotoxin.^12–14 In 1990s, third-generation immunotoxins were made by recombinant DNA technology which include Fv fragment of antibodies and were called recombinant immunotoxins.¹⁵ Recombinant immunotoxins were homogeneous and cost effective. In the last efforts, fusion proteins including antibody fragments and enzymatically active toxin domains were produced by molecular cloning techniques.¹⁶ The cytotoxic part of the newly designed immunotoxins was an endogenous protein of human source similar to proapoptotic proteins or RNase.¹⁷

DAB389IL2 was the first immunotoxin approved by the Food and Drug Administration (FDA), well known as denileukin diftitox (DD; Ontak^®).¹⁸ Immunotoxins’ use against cancer cells may be useful as part of a combined treatment with other agents, but on the other hand, they may be effective clinically as an agent targeting circulating tumor cells expressing a high number of target antigens. Currently, the process of finding agents that work well in combination with immunotoxins is ongoing. However, the most valuable compounds should have minimal immunogenicity and the most potency for target cell killing.¹⁹

Toxin

Protein toxins first were known as pathogenic factors released by bacteria or poisonous plants.²⁰ They have enzymatic activity at very low concentrations on the target cells and result in cell death.^21,22 Exogenous proteins have many benefits but they can be limited by neutralizing activity of antidrug antibodies, shortening half-life in the circulation and immune responses.^23,24 The toxins used in immunotoxin constructs are very potent and produced by microorganisms, plants, insects, and vertebrates. Immunotoxins contain several domains. A cell-binding domain attaches the toxin to the cancer cell surface, a translocation domain could help the toxin to pass through membrane into the cytosol, and a catalytic domain (death domain) kills the cells or inactivates vital cellular process.^16,25 Till now, reported immunotoxins have been constructed using the toxins saporin, mistletoe lectin-1, gelonin, pokeweed antiviral protein (PAP), and ricin from plant sources and shiga toxin, DT, and PE from bacterial sources.^16,26,27 PE and DT have very different amino acid sequences; the catalytic domain of PE is located in the carboxyl terminus, but in DT, it is near the amino terminus. In contrast, the binding domain of PE is located in amino terminus, and in DT, it is near the carboxyl terminus.²⁸

PE is a single polypeptide chain with size of 66 kDa, containing 613 amino acids and three functional domains that kill mammalian cells by binding to surface receptor, internalization via coated pits, and finally translocation of an enzymatically active C-terminal fragment to the cytosol. This fragment inhibits protein synthesis by adenosine diphosphate (ADP) ribosylation of elongation factor 2.²⁹ DT is a single-chain protein with 535 amino acids containing an enzymatic A domain and a binding B domain.³⁰ In contrast to plant toxins, the bacterial toxins transfer ADP ribose from nicotinamide adenine dinucleotide (NAD) to diphthamide (modified histidine) and inactivate elongation factor-2 and inhibit protein synthesis.³¹ However, the plant toxins (e.g. dianthin, saporin, and gelonin) are N-glycosidase, which hydrolyses the 28S-rRNA and inactivates the 60S subunit in eukaryotic ribosomes.^32,33 The N-glycosidase enzymes are called ribosome-inactivating proteins (RIPs)^9,34 that according to their structure and function separated into two subgroups: type-1 RIPs (hemitoxins) have only a catalytic domain and have a lower cytotoxicity because they enter cells in an unspecific way.^35,36 Type-2 RIPs (holotoxins) such as ricin that was part of the first immunotoxins have a catalytic and a receptor-binding domain.³⁷

Ricin a heterodimeric protein (64 kDa) is an extremely potent toxin isolated from castor beans, and it can be classified as a possible biological weapon. Ricin and other plant lectins, such as abrin and modeccin, consist of an A-chain that damages ribosomes and suppresses protein synthesis, and a B-chain that (binding domain) play role for cellular uptake. According to recent studies, ricin not only inhibits protein synthesis but also induces apoptosis in different cells. However, the relationship between protein synthesis inhibition and apoptosis induction was not clear in this study.

The potent toxin, abrin, extracted from Abrus precatorius seeds, is a type-2 RIP, and it has the most catalytic efficiency among toxins of this class, but in targeted therapy, it is not used much.⁵

Toxin resistance

Until now, three kinds of toxin resistance are known: cellular, organismal, and immunologic. Cellular resistance is due to defect in delivery of toxin to the cytosol^37–40 and organismal resistance is because of failure to form ADPr EF2⁴¹ or poor starting of apoptosis. Resistance to apoptosis is a major obstacle to effective cytotoxic cancer therapy.⁴² Finally, immunologic resistance occurs when patients with healthy immune system produce neutralizing antibodies against an immunotoxin.⁴³ According to the increased application of these proteins as clinical therapeutics, the issue of immunogenicity becomes highlighted.¹

Antibody

Use of monoclonal antibody (mAb) is a new evolution to cancer treatment. Several mAbs are used in treatment of some kinds of cancer which are very effective. Up to now, 28 different drugs have been approved for cancer therapy by FDA, USA.⁴⁴ But, many types of cancer are naturally resistant to mAbs. In this condition, if a cytotoxic agent attaches to the antibody, it can affect cancerous cells.

Immunoconjugates are made from a chemotherapy drug, radioisotope, or toxin which is attached to an antibody.^16,45 The selection of the antibody is one of the important factors in immunotoxin design. A number of antibodies do not have an effect on killing of cancer cells by itself, but they can be useful for delivery of cytotoxic agents into cancer cells as immune factors. The antibodies recognize an antigen that is presented on the cancer cell surface, and antibody binds to toxin and enters into the endocytic compartment. The targeted antigen must not be present on the surface of normal cells and not be readily poured into the circulatory system.¹⁶ Antibodies were conjugated through a disulfide chemical bond either to the holotoxins or to their catalytic subunits. Reduction can remove each part from its binding domain.⁴⁶ Recently, active single-chain Fv fragments of antibodies have been cloned in Escherichia coli by attaching the light- and heavy-chain variable domains together with a peptide linker.^47,48

Fv fragment is the smallest binding unit of an antibody which consists of a light- and heavy-chain variable domain, and both chains are linked together with flexible peptide linkers. Using this small fragment increases the efficiency of penetrating into the tumor cell¹⁶ (Figure 1). Nowadays, segments of recombinant antibodies (RITs) become a common part of immunotoxin designation. Because RITs are not human proteins, immunogenic activities can be an obstacle to their development.¹ The Fv adjoins to tumor-associated antigens (e.g. CD22, CD25, mesothelin, and IL-3 receptor) along with the bacterial toxin, a 38 kDa fragment of Pseudomonas exotoxin A (PE38), DT, or ricin in order to specifically target and kill cancer cells, which is followed by endocytosis. After proteolytic degradation, a fragment of PE38 catalyzes the ADP ribosylation and inactivation of EF2 when imported to the cytosol which resulted in protein synthesis inhibition and led to the cell death.^49–52

Figure 1.

Fab, Fc, and Fv portions of antibody molecule. IgG molecules are divided into several functional domains. The main portions are Fc and Fab. The Fab fragment is further divided into the Fv fragment, the smallest fragment that retains antigen binding via contact with both the heavy and light chains. The two chains of the Fab fragment are held together by a naturally occurring disulfide bond. There is not such covalent bond in Fv. The two chains of Fv are held together either by a flexible peptide linker or by a novel disulfide bond.

Immunotoxin

Classically, immunotoxins (ITs) are bifunctional chimeric molecules composed of an antibody fragment linked to a toxin component. Immunotoxins receive their toxic potency from the toxin and their specificity from the antibody. It is necessary to clarify that none of all immunotoxins use antibodies for specific targeting; instead, they use smaller molecules such as growth factors and different cytokines. Historically, immunotoxins can be divided into three evolutionary groups. The first group was constructed by connecting whole toxins to antibodies using chemical reagents that form covalent disulfide bonds. Because of the existence of the native toxin in immunotoxin structure, these molecules target normal cells as well. In the second group of immunotoxins, cell-binding domains of toxin were deleted. Like first group, the second group was produced by chemical methods. Although the second group of immunotoxins did not recognize normal cells, they had problems such as having a large molecular size and immunologic side effects. In third group of immunotoxins, recombinant DNA technology overcomes these problems. Recombinant immunotoxins are made of variable fragment (Fv) of heavy and light chains of the antibodies combined to functional component of toxins lacking cell-binding domains. Recent challenge in the field is elimination of immunogenicity of the toxin to human immune system, introducing the next immunotoxin group as humanized immunotoxins (Figure 2).¹⁷

Figure 2.

Different generations of immunotoxins. (a) Whole toxin of diphtheria exotoxin structure with related functional domains. (b) The first-generation immunotoxins were constructed from intact toxins and intact antibodies attached using chemical crosslinking agents. (c) Second-generation immunotoxins used modified toxins lacking receptor-binding domains. (d) Third-generation immunotoxin molecules were made from cloned antibody fragments fused to a genetically modified toxin.

Immunology of immunotoxin

Immunotoxin as an immunogenic agent

The human immune system protects body against any abnormal situation provided by invading organisms as well as cancer. This property is concluded from specific discrimination of self from non-self molecules by compromising immune system. Entry of any foreign molecule into body elicits immune system to eliminate the invader. Immunotoxin as a molecule that is composed of bacterial or plant toxin and murine antibody is a noticeable target for immune system. When immunotoxin, as a foreign protein, enters into body, it stimulates intact immune system by generating human anti-mouse antibody (HAMA) and human anti-toxin antibody (HATA). These neutralizing antibodies recognize immunotoxin molecules at next exposure and make them inactivated. Immunogenicity of immunotoxin is the most important limiting factor when used for therapeutic purposes and an obstacle in the development of classic immunotoxins. For overcoming this problem, the immunogenicity of immunotoxin must be decreased as much as possible.^53–55 Hence, modification of immunotoxin molecular structure should be done in both antibody and toxin aspects.

Engineered antibodies

Most monoclonal antibodies (mAbs) that were used in the construction of the first and second groups of immunotoxins have murine origin.⁵⁶ Antigenic differences among human and mouse antibodies known as isotopic determinants, especially in constant fragment (Fc) region, lead to undesirable immune response. Third group of immunotoxins are made by recombinant DNA techniques and use Fv fragments of antibody.⁴⁴ Modification of Fv fragments in order to perfectly attach to the toxin partner creates recombinant immunotoxins with the lowest side effects. Antibody engineering technology helps us to select the best peptide regarding molecular conformation, immunogenicity, and antigen recognition.⁵⁷ Since the first report of Fv production in 1988, the number of developed third-generation immunotoxins has passed 1000.⁴⁸

Modified toxins

Bacterial and plant toxins as well as murine antibodies used in the development of immunotoxins are highly immunogenic to human. In order to overcome this problem, various strategies have been tried. Notably, among them is site-directed mutagenesis of toxin genes to generate less immunogenic and more stable variants. Tangri et al.⁵⁸ by modification of helper T-cell epitopes within two regions of erythropoietin reduced in vitro immunogenicity of two modified forms of erythropoietin with no change in bioactivity. Onda et al. produced a less immunogenic immunotoxin by identifying and eliminating most of the B-cell epitopes on Pseudomonas exotoxin A (PE38). This is possible by substitution of specific large hydrophilic amino acids with originals. The last immunotoxin was significantly less immunogenic in three strains of mice, yet retains full cytotoxicity and anti-tumor activities.^57–59

Humanized immunotoxins

Due to limited success achieved with modified toxins in animal models and clinical studies, efforts are in progress to replace cytotoxic part of immunotoxin with endogenous protein of human origin (e.g. proapoptotic protein). In this way, new generation of immunotoxins called humanized immunotoxins are made. Dälken et al. employed human granzyme B (GrB) as an effector in chimeric fusion proteins which also contains the epidermal growth factor receptor (EGFR) ligand, transforming growth factor-alpha (TGF-α), or an ErbB2-specific single-chain antibody fragment (scFv) which can target tumor cells selectively. Treatment with picomolar to nanomolar concentrations of synthetic proteins resulted in selective and rapid tumor cell killing (i.e. apoptosis).⁶⁰ Liu et al. designed a recombinant protein, GrB/scFvMEL, including human granzyme B (GrB) fused to the single-chain antibody scFvMEL. This immunotoxin induces apoptosis with targeting melanoma gp240 antigen.⁶¹ Bremer et al. fused the antibody fragment scFvC54 with human soluble tumor necrosis factor–related apoptosis-inducing ligand (sTRAIL). This protein made a crosslink with agonistic TRAIL receptors and induced apoptosis through specific binding of scFvC54:sTRAIL to the epithelial glycoprotein 2 (EGP2) (highly expressed carcinoma-associated cell surface antigen). Neighboring tumor cells that were devoid of EGP2 expression (bystander cells) experienced a proapoptotic effect as a result of scFvC54:sTRAIL activities.^62–64 Psarras et al. introduced a human immunotoxin analogue that was produced in E. coli by fusing the human pancreatic RNase1 (hpRNase1) gene encoding with that of human interleukin-2 (hIL-2). The recombinant hpRNase1-hIL-2 inhibited protein synthesis in HTLV-1-infected malignant T-cells which produce increased level of high-affinity IL-2 receptors (IL-2R).⁶⁵ An immune-RNase was reported by De Lorenzo et al.⁶⁶ which is made up of a scFv linked to the most specific tumor-associated antigen ErbB2. ErbB2 overexpresses on tumor cells of different origin and human ribonuclease and binds specifically to ErbB2-positive cells selectively kills these cells. Mathew et al.⁶⁷ generated a novel humanized chimeric protein with human DFF40 fused with granulocyte-macrophage colony-stimulating factor (GMCSF) to target acute myeloid leukemia (AML) cells by inducing apoptosis. Zhou et al. provided a recombinant protein containing a humanized dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa (Fn14) antibody fused with recombinant gelonin toxin as a potential therapeutic agent (called hSGZ). The hSGZ immunotoxin is highly potent and selectively kills Fn14-positive tumor cells (e.g. lung, melanoma, and breast cancers) in vitro.⁶⁸

Application of immunotoxins

Immunotoxins in cancer therapy

Targeted therapy is an emerging method in cancer therapy and is going to replace the conventional therapies in future. For effective treatment of cancer, it is necessary to specifically direct the killing agent toward the surface molecules of tumor cells. Immunotoxin molecules are highly potent agents in cancer therapy as they contain selective binding domains.⁶⁹

Targeting hematological tumors

A wide range of hematological cancers, from leukemia to multiple myeloma, have been challenged with different immunotoxins. Clinical studies were done on hematological tumors listed in Table 1. Major features of Ontak, the first FDA-approved immunotoxin, are described later.¹³

Table 1.

Immunotoxins targeted hematological tumors.

Immunotoxin	Target	Targeting moiety	Toxic moiety	Tumor type	Reference
DAB389IL2	IL-2R	IL-2	Diphtheria toxin (DAB389)	CTCL, NHL, CLL, NSCLC, melanoma, ovarian, and breast cancers	Foss et al.⁷⁰, Frankel et al.,⁷¹ and Dang et al.⁷²
BL22	CD22	Anti-CD22 dsFv	PE38	Cladribine-resistant HCL	Kreitman et al.⁷³
BL22 and RFB4-PE38	CD22	Anti-CD22 dsFv	PE38	HCL, CLL, and ALL	Herrera et al.⁷⁴ and Schindler et al.⁷⁵
HA22	CD22	Anti-CD22 dsFv	PE38	HCL, ALL, NHL, and CLL
DT388 GMCSF	GMCSFR	GMCSF	Diphtheria toxin (DAB388)	AML	Frankel et al.⁷⁶
RFB4-dgA	CD22	Anti-CD22 Fab′	Deglycosylated RTA	B-NHL	Kreitman et al.⁷³
HD37-dgA	CD19	Anti-CD19 Mab	Deglycosylated RTA	NHL	Sandler et al.⁷⁷
Combotox (RFB4-dgA + HD37-dgA)	CD19, CD22	Anti-CD22 Fab′ + Anti-CD19 Mab	Deglycosylated RTA	NHL and ALL	Herrera et al.⁷⁴
RFT5-dgA (IMTOX-25)	CD25	Anti-CD25 Mab	Deglycosylated RTA	HD, CTCL, and melanoma	Prince et al.⁴⁹
Ki-4.dgA	CD30	Anti-CD30 Mab	Deglycosylated RTA	HD and NHL	Okeley et al.⁷⁸ and Younes et al.⁷⁹
HuM195/rGel	CD33	Anti-CD33 Mab	Gelonin	AML and CML	Löwenberg et al.⁸⁰ and Aplenc et al.⁸¹

CTCL: cutaneous T-cell lymphoma; NHL: non-Hodgkin lymphoma; CLL: chronic lymphocytic leukemia; NSCLC: non-small-cell lung carcinoma; HCL: hairy cell leukemia; ALL: acute lymphocytic leukemia; AML: acute myelogenous leukemia; B-NHL: B-cell NHL; HD: Hodgkin’s disease; dgA: deglycosylated ricin A-chain; RTA: ricin A-chain; GMCSF: granulocyte-macrophage colony-stimulating factor; rGel: recombinant gelonin; MAb: monoclonal antibody; scFv: single-chain variable fragment; dsFv: disulfide-stabilized Fv antibody fragment; GMCSFR: granulocyte-macrophage colony-stimulating factor receptor.

DD (Ontak™)

DD or DAB389IL2 (Ontak™) is the first immunotoxin that FDA approved for clinical usages. It is used in the treatment of recurrent cutaneous T-cell lymphoma (CTCL). It is the chimeric protein of human IL-2 and a truncated form of DT (DAB389). Ontak targets the high-affinity IL-2R, which is overexpressed in various tumors such as CTCL, adult T-cell leukemia (ATL), Hodgkin’s disease (HD), and other B- and T-cell leukemias and lymphomas. Also, it has a lower expression on normal activated T-cells and T-regulatory cells. Despite retreatment limitation and significant side effects of usage, Ontak is being currently evaluated in combination with other treatments for various cancers.⁸²

Targeting solid tumors

Application of immunotoxins in solid tumor treatment encounters some obstacles. Ineffective penetration of immunotoxins into tumor tissues and neutralizing activity of perfect immunity of patients affected development of immunotoxins. Emerging new technologies would help us to refine immunotoxin designing to defeat such problems. Among the immunotoxin therapies against solid tumors, treatment of brain tumors has had a promising result because of emerging new drug delivery methods, thereby facilitating immunotoxin penetration.⁸³ Clinical studies on solid tumors have been listed in Table 2.

Table 2.

Immunotoxins targeted solid tumors.

Immunotoxin	Target	Targeting moiety	Toxic moiety	Tumor type	Reference
Erb38	erbB2/HER2	Anti-Her2/neu dsFv	PE38	Breast carcinoma	Choudhary et al.⁸²
scFv(FRP5)-ETA	erbB2/HER2	Anti-Her2/neu scFv(FRP5)	ETA	Melanoma, breast, and colon	Choudhary et al.⁸²
SS1P (SS1(dsFv)-PE38)	Mesothelin	Antimesothelin dsFv	PE38	Mesothelioma, ovarian, and pancreatic cancers	Hassan et al.⁸⁴
LMB-1	Lewis Y	Anti-Lewis Y MAb	PE38	Adenocarcinoma	Pastan et al.⁴⁵
LMB-7	Lewis Y	Anti-Lewis Y scFv(B3)	PE38	Adenocarcinoma	Pastan et al.⁴⁵
LMB-9	Lewis Y	Anti-Lewis Y dsFv(B3)	PE38	Adenocarcinoma	Pastan et al.¹³
SGN-10	Lewis Y	Anti-Lewis Y dsFv(BR96)	PE40	Adenocarcinoma	Posey et al.⁴³
OvB3-PE	Ovarian cancer	Murine MAb	PE	Ovarian carcinoma	Pai et al.⁸⁵
TP40	EGFR	TGF-α	Modified PE40	Bladder cancer	Messing and Reznikoff ⁸⁶

EGFR: epidermal growth factor receptor; TGF: transforming growth factor.

Immunotoxins in autoimmune disorders

Targeting surface markers on specific cells is promising in treatment of autoimmune diseases. Most of efforts were done in preclinical level and need to be completed.⁷⁴ Application of anti-CD64-ricin immunotoxin for clearing of activated synovial macrophages in rheumatoid arthritis was effective in vitro.⁸⁷ Stepanov and co-workers evaluated in vitro a panel of immunotoxins using the B-cell target moiety c-myc epitope.⁸⁸ Similar to hematological tumors, Ontak^™ showed promising results in clinical trials of psoriasis and rheumatoid arthritis treatment.⁸⁹

Clinical trials

At the early 1970s, for the first time, it is found that immunotoxins are potential cancer cell killers. Initially, in primary clinical studies, topical forms of refractory metastatic cancers were directed with direct injection of toxins with no modification.⁹⁰ The ability of immunotoxins in the treatment of a wide variety of hematologic malignancies was tested in clinical trials. There are some records that documented the benefits of immunotoxins in the treatment of tumors with or without peripheral blood involvement (e.g. leukemia, Hodgkin’s lymphoma and multiple myeloma).⁹¹

Regarding periods of development, various generations of immunotoxins which were clinically evaluated can be distinguished easily. It has been found that first generation of immunotoxins (e.g. OVB3-PE and 260F9-rRTA) often cause severe side effects that, in many cases, are not followed in the late stages of clinical treatment.⁹² LMB-2 was evaluated in a phase-I study completed in 2011 and applied in patients with hematologic malignancies; of 35 patients, complete response (CR) has been found in 1 patient, while partial response (PR) showed in 7 patients with a total response rate of 24%.⁹² In 2001, initial approval of DD for treatment of cutaneous T-cell lymphoma (CTCL) was granted by the FDA. DD contains a traditional DT backbone; however, the targeting domain of DD does not contain an antibody. Instead, a recombinant human IL-2 is fused to the first 388 amino acids of DT at C-terminus of the toxin.⁹³ The ligand targets cells that express IL-2R which persistently expresses in a number of hematologic malignancies such as CTCL which makes it a good therapeutic target. Testing of DD in a single-arm phase-III trial of patients with recurrent IL-2-positive CTCL demonstrated a response rate of 30% with a median response duration of 6.9 months.⁹⁴ A later randomized, phase-III, placebo-controlled trial of DD for patients with CTCL confirmed improved overall response rate and progression-free survival with an acceptable safety profile in patients with early- and late-stage CTCL.⁴⁹ In practice, DD is not used frequently because of poor tolerability and some side effects such as flu-like symptoms and VLS. A number of recent investigated immunotoxins and those that are in ongoing clinical trials are listed in Table 3. Table 4 presents other promising immunotoxins in preclinical setting, which may be shortly tested.

Table 3.

List of some immunotoxins which have been investigated in recent or ongoing clinical trials.

Immunotoxin	Target identifier/payload	Regimen	Patient population	Phase	Trial identifier	Year
Denileukin diftitox	IL-2R/DT	IV single agent	Cutaneous T-cell lymphoma	NA	NA open trials
Moxetumomab pasudotox	CD22/PE	IV single agentIV single agentIV single agent	Hairy cell leukemiaAdult ALLChildhood ALL or non-Hodgkin’s	IIII/IIII	NCT01829711NCT01891981NCT00659425	201520132015
LMB-2	CD25/PE	IV single agentIV with fludarabine/cyclophosphamide	Hairy cell leukemiaAdult T-cell leukemia	IIII	NCT00321555NCT00924170	20062009
A-dmDT390-bisfv (UCHT1)	CD3ϵ/DT	IV single agent	Cutaneous T-cell lymphoma	I/II	NCT00611208	2008
DT2219ARL	DT19/CD22/DT	IV single agent	B-cell lineage leukemia or lymphoma	I	NCT00889408	2009
SS1P	MSLN/PE	IV with pentostatin/cyclophosphamide	Mesothelioma	II	Completed trials
MOC31PE	EpCAM/PE	IV single agent	Epithelial carcinomas	I	NCT01061645	2010
Oportuzumab monatox	EpCAM/PE	–	Bladder cancer	In situ	Not listed
HuM195-gelonin	CD33/gelonin	IV single agent	AML, CML, and MDS	I	NCT00038051	2002

ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; CML: chronic myelogenous leukemia; DT: diphtheria toxin; IL-2R: interleukin-2 receptor; IV: intravenous; MDS: myelodysplastic syndrome; MSLN: mesothelin; NA: not applicable; PE: Pseudomonas exotoxin A.

Table 4.

Immunotoxins under preclinical testing.

Immunotoxin	Target	Payload	Disease	Advance	Year
RG7787	MSLN	PE	Mesothelioma, pancreatic, decreased immunogenicity cholangiocarcinoma, gastric, TNBC, and ovarian	Decreased immunogenicity and toxicity	2014
VB8-845	EpCAM	DeBouganin	Epithelial carcinoma	Less immunogenic	2012
hSGZ	fn14	Gelonin	Lung, melanoma, and breast	New target	2013
D2c7-(scdsfv)-PE38KDEl	EGFR and PE	Binds both EGFRwt and EGFRvIII	Glioblastoma multiforme	EGFRvIII	2013

EGFR: endothelial growth factor receptor; EGFRvIII: endothelial growth factor receptor variant III; MSLN: mesothelin; PE: Pseudomonas exotoxin A; TNBC: triple-negative breast cancer.

Conclusion

Immunotoxins are high-capacity anti-tumor agents that selectively targeted the surface antigens’ expression on tumor cells. Immunotoxins are very useful in the treatment of circulating tumor cells expressing a high number of target proteins. Yet, however, there are some problems in applying immunotoxins alone in cancer treatment; for example, because of the low-level expression of surface antigens on non-circulating tumors, normal cells may affect. Hence, immunotoxins could be helpful as part of a combined agent with other treatments (e.g. radiotherapy).

Recent advances in genetic engineering and attempts to find new tumor markers—which are confined to cancer cells—pave the way for developing immunotoxins capable of killing target cells with high specificity while having lower toxicity and immunogenicity.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Immunotoxin: A new tool for cancer therapy

Abstract

Keywords

Introduction

History

Toxin

Toxin resistance

Antibody

Immunotoxin

Immunology of immunotoxin

Immunotoxin as an immunogenic agent

Engineered antibodies

Modified toxins

Humanized immunotoxins

Application of immunotoxins

Immunotoxins in cancer therapy

Targeting hematological tumors

DD (Ontak™)

Targeting solid tumors

Immunotoxins in autoimmune disorders

Clinical trials

Conclusion

Footnotes

Declaration of conflicting interests

Funding

References